PROJECT SUMMARY Lymphocyte development is precisely controlled to enable clonal expansion and generation of a diverse immunoglobulin receptor repertoire, which proceeds through DNA double-stranded breaks (DSBs). These two dichotomous, but interdependent processes, are managed through the cooperation of diverse cellular signals to prevent cells with DSBs from entering cell cycle where they could be aberrantly repaired as translocations. During early B cell development, the pre-B cell receptor (pre-BCR), through activation of the SYK kinase, coordinates both the proliferative expansion of pre-B cells and the assembly of immunoglobulin receptor genes. Negative regulation of the pre-BCR is required to enforce cell cycle arrest and limit the number of DNA breaks generated during immunoglobulin receptor gene assembly. Indeed, unopposed pre-BCR signaling, particularly increased SYK activity, drives proliferation and leukemic transformation. We have identified a novel cell-type specific checkpoint pathway activated by signals from DSBs that inhibits pre-BCR signaling. The physiologic DSBs generated during immunoglobulin receptor gene rearrangement trigger DNA damage responses that suppress SYK kinase activity downstream of the pre-BCR. Surprisingly, this signaling network is not triggered by genotoxic DNA injury and, thus, is specific to the DSBs generated during normal B cell differentiation. In early B cells, distinct cellular responses to physiologic and genotoxic DSBs are essential for ensuring normal B cell differentiation and inhibiting leukemic transformation. Our goal is to determine how signals from DSBs integrate with developmental programs to coordinate B cell maturation. We propose that early B cells toggle between signals from the pre-BCR and DSBs to order immunoglobulin receptor gene assembly and maintain genomic stability by preventing proliferation of cells with DSBs. Utilizing an innovative experimental approach that permits isolation of DSB signals and surface receptor signals, we propose to: 1) define the transcriptional repressor complex that regulates DSB-mediated checkpoints, 2) identify the DSB-dependent post-translational pathways that suppress pre-BCR signaling, and 3) determine the DSB-specific pathways that dictate the unique cellular responses to physiologic versus genotoxic insults. Completion of these studies will delineate a set of mechanisms critical for dampening pre-BCR signals, will establish novel paradigms for cell- type specific checkpoints to DSBs, and will define the underlying principles for discrete cellular responses to physiologic versus genotoxic DSBs.